This work has been supported by the German Research Foundation (DFG) SCHU-2681/1-1 (C.S.) and KFO 146 (M.F.), the DOD Department of Defence grant PC 040972 (M.O.) and the National Institute of Health grants RO1 CA108835, RO1 Ai44129 (J.P.S.)

1. Generation of HLA-A2-Ig Based KaAPC
<ol>
	<li>Prepare sterile borate buffer. Make a 0.1 M solution of boric acid in water and adjust the pH to 7.0. Sterile filter (0.22 &#956;m) buffer and store at 4 &#176;C.</li>
	<li>Prepare sterile bead wash buffer. Use phosphate buffered saline (PBS) without magnesium and calcium. Add human AB serum for 3% final concentration. Add ethylenediaminetetraacetic acid (EDTA) for 2 mM final concentration. Add sodium azide (NaN3) for 0.01% final concentration. Sterile filter (0.22 &#956;m) buffer and store at 4 &#176;C.</li>
	<li>Transfer 250 &#956;l of epoxy beads (about 100 x 106 beads) from stock into a sterile, screw top glass vial.</li>
	<li>Add 250 &#956;l of borate buffer.</li>
	<li>Put glass vial on a magnet and let beads adhere for 2 min; aspirate buffer and remove glass vial from magnet.</li>
	<li>Wash beads once with 1 ml borate buffer.</li>
	<li>Resuspend beads into 550 &#956;l of 0.018 &#956;g/ml HLA-A2-Ig and 3.64 &#956;g/ml anti-human CD95 mAb (clone CH11) and gently mix by shaking. (Please see discussion section for additional information.)</li>
	<li>Incubate glass vial on a rotator end over end, at 4 &#176;C for 24 hr.</li>
	<li>Spin down beads at 300 x g for 2 min.</li>
	<li>Place glass vial onto a magnet for 2 min and aspirate the &#8220;KaAPC loading solution&#8221;; remove glass vial from magnet, add 1 ml of bead wash buffer and resuspend carefully by shaking.</li>
	<li>Repeat step 1.10 twice.</li>
	<li>Incubate glass vial on a rotator at 4 &#176;C for 24 hr in 1 ml bead wash buffer. At this point, all residual protein binding sites will be blocked by human AB serum contained in the bead wash buffer.</li>
	<li>Spin down beads at 300 x g for 2 min.</li>
	<li>Place glass vial onto a magnet for 2 min and aspirate bead wash buffer; remove glass vial from magnet and replace solution with 1 ml of PBS.</li>
</ol>
2. Quality Control, Peptide Loading, Washing, Storage, and Stability of KaAPC
<ol>
	<li>Transfer 1.5 x 105 KaAPC into a FACS tube and wash with 1 ml FACS wash buffer.</li>
	<li>Resuspend beads in 100 &#956;l FACS wash buffer and add 1 &#956;l of anti-mouse IgG1 PE (for detection of the Fc part of the HLA-A2-Ig) and 1 &#956;l of anti-mouse IgM FITC (clone R6-60.2, for detection of the anti-human CD95 mAb).</li>
	<li>Incubate for 15 min at 4 &#176;C, wash with 1 ml FACS wash buffer, and resuspend in 250 &#956;l FACS wash buffer and read staining by flow cytometry.</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51859/51859fig1highres.jpg" width="500" /> 
Figure 1. QC stain of a KaAPC batch made as stated in the protocol section. Staining was performed as described in protocol section 2. HLA-A2-Ig was detected using an anti-mouse-IgG1 PE mAb, whereas anti-human-CD95 (anti-Fas) was detected with an anti-mouse-IgM FITC mAb (see protocol section 2.2) (blue line). Filled black graphs represent empty beads stained with anti-mouse-IgG1 PE mAb or anti-mouse-IgM FITC mAb, respectively. Numbers in the upper right corner indicate the mean fluorescent intensity (MFI). <a href="https://www.jove.com/files/ftp_upload/51859/51859fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>For peptide loading transfer 20 x 106 KaAPC into a new sterile glass vial, place onto a magnet for 2 min, aspirate PBS and resuspend beads into 500 &#956;l fresh PBS with 5 &#956;g peptide (Keep in mind, only HLA-A2 restricted peptides will bind onto the dimer!).</li>
	<li>Store loaded KaAPC at 4 &#176;C until use, for at least 3 days to allow sufficient peptide loading onto the HLA-A2-Ig dimer.</li>
	<li>Immediately prior to use, wash the peptide loaded KaAPC as indicated in 1.14; repeat this step at least 6x. 
	NOTE: This is a crucial step to ensure that free peptide has to be removed completely to avoid false positive killing results; residual free peptide has been shown to induce apoptosis in antigen-specific T cell cultures9. To rule out a soluble peptide effect, run supernatant control samples of peptide loaded and washed KaAPC (see 3.8).</li>
	<li>Count beads using hemocytometer and label with date, name of peptide and concentration. 
	NOTE: Loaded KaAPC remain functional at 4 &#176;C for at least one month. After one month, re-loading with the same peptide allows KaAPC to be utilized for up to one year.</li>
</ol>
3. KaAPC In Vitro Killing Assay
<ol>
	<li>Prepare co-culture media. Prepare complete RPMI medium supplemented with 3% T cell growth factor (made in the lab6) and 3% donor autologous plasma. If donor autologous plasma is unavailable, use heat-inactivated human AB serum.</li>
	<li>Prepare AnnexinV binding buffer preparation. Dissolve 8.12 g sodium chloride (NaCl) and 0.12 g calcium chloride (CaCl2) into 990 ml ddH2O.Add 10 ml 1M HEPES buffer.</li>
	<li>Generate human antigen-specific T cells. (For a detailed protocol please see a previous publication7 utilizing artificial Antigen Presenting Cells (aAPC)). Ensure that peptide specificity is at least &#62;75% as determined by tetramer staining.</li>
	<li>For each sample incubate 2 x 105 antigen-specific T cells/well (in a 96 well round bottom plate) in 200 &#956;l of co-culture medium at a 1:1 ratio with KaAPC for 48 hr in a humidified incubator at 37 &#176;C and 5% CO2.</li>
	<li>Harvest samples and transfer into a FACS tube; wash with 1 ml AnnexinV binding buffer, spin down at 300 x g for 5 min, discard supernatant and resuspend in 100 &#956;l of AnnexinV binding buffer</li>
	<li>Stain samples with 1 &#956;l AnnexinV FITC and 5 &#956;l 7-AAD for 10-15 min at RT, wash with 1 ml AnnexinV binding buffer; spin down at 300 x g for 5 min, discard supernatant and resuspend in 250 &#956;l AnnexinV binding buffer</li>
	<li>Read samples immediately on flow cytometer.</li>
	<li>Prepare the following samples to determine antigen-specific killing by KaAPC: T cells only; T cells + soluble anti-CD95; T cells + unloaded KaAPC; T cells + non-cognate peptide loaded KaAPC; T cells + cognate peptide loaded KaAPC; T cells + supernatant of cognate peptide loaded KaAPC.</li>
</ol>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51859/51859fig2highres.jpg" width="500" /> 
Figure 2. KaAPC killing assay. Peptide loaded KaAPC or control beads (HLA-A2-Ig only beads) were cultured for 48 hr at a 1:1 ratio with CMVpp65 specific T cells (~80%), harvested and subsequently stained with AnnexinV and propidium iodide (PI) to determine apoptosis by flow cytometry (see protocol section 3.3-3.7). &#8220;cognate&#8221; refers to beads loaded with CMVpp65 and &#8220;non-cognate&#8221; to beads loaded with Mart-1 peptide. Numbers indicate the percentage of viable T cells in the lower left quadrant of each plot. <a href="https://www.jove.com/files/ftp_upload/51859/51859fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="9">
	<li>To perform crisscross-experiments, generate antigen-specific T cells of two different specificities.</li>
	<li>Further evaluate the antigen-specific depletion-abilities of KaAPC with a simultaneous analysis of the antigen&#8211;specific elimination of T cells in heterogeneous T cell population. (For a detailed protocol please see Sch&#252;tz&#160;et al.10).</li>
</ol>

<ol><li>Bizzaro, N., Tozzoli, R., Shoenfeld, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Are+we+at+a+stage+to+predict+autoimmune+rheumatic+diseases%5BTitle%5D)+AND+%22Arthritis+and+rheumatism%22%5BJournal%5D)">Are we at a stage to predict autoimmune rheumatic diseases.</a> Arthritis and rheumatism. 56 (6), 1736-1744 (2007).</li>
<li>Schütz, C., Oelke, M., Schneck, J. P., Mackensen, A., Killer Fleck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((artificial+antigen-presenting+cells%3A+the+synthetic+embodiment+of+a+%E2%80%9Cguided+missile%5BTitle%5D)+AND+%22Immunotherapy%22%5BJournal%5D)">artificial antigen-presenting cells: the synthetic embodiment of a “guided missile.</a> Immunotherapy. 2 (4), 539-550 (2010).</li>
<li>Hoves, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mature+but+not+immature+Fas+ligand+%28CD95L%29-transduced+human+monocyte-derived+dendritic+cells+are+protected+from+Fas-mediated+apoptosis+and+can+be+used+as+killer+APC%5BTitle%5D)+AND+%22Journal+of+immunology+%28Baltimore%2C+Md.%3A+1950%29%22%5BJournal%5D)">Mature but not immature Fas ligand (CD95L)-transduced human monocyte-derived dendritic cells are protected from Fas-mediated apoptosis and can be used as killer APC.</a> Journal of immunology (Baltimore, Md.: 1950). 170 (11), At 
 <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed/12759415">http://www.ncbi.nlm.nih.gov/pubmed/12759415</a> 5406-5413 (2003).</li>
<li>Hoves, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elimination+of+activated+but+not+resting+primary+human+CD4%2B+and+CD8%2B+T+cells+by+Fas+ligand+%28FasL%2FCD95L%29-expressing+Killer-dendritic+cells%5BTitle%5D)+AND+%22Immunobiology%22%5BJournal%5D)">Elimination of activated but not resting primary human CD4+ and CD8+ T cells by Fas ligand (FasL/CD95L)-expressing Killer-dendritic cells.</a> Immunobiology. 208 (5), 463-475 (2004).</li>
<li>Schütz, C., Hoves, S., Halbritter, D., Zhang, H. -G., Mountz, J. D., Fleck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alloantigen+specific+deletion+of+primary+human+T+cells+by+Fas+ligand+%28CD95L%29-transduced+monocyte-derived+killer-dendritic+cells%5BTitle%5D)+AND+%22Immunology%22%5BJournal%5D)">Alloantigen specific deletion of primary human T cells by Fas ligand (CD95L)-transduced monocyte-derived killer-dendritic cells.</a> Immunology. 133 (1), 115-122 (2011).</li>
<li>Oelke, M., Maus, M. V., Didiano, D., June, C. H., Mackensen, A., Schneck, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+induction+and+expansion+of+antigen-specific+cytotoxic+T+cells+by+HLA-Ig-coated+artificial+antigen-presenting+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells.</a> Nature. 9 (5), 619-624 (2003).</li>
<li>Chiu, Y. -L., Schneck, J. P., Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HLA-Ig+based+artificial+antigen+presenting+cells+for+efficient+ex+vivo+expansion+of+human+CTL%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments+%3A+JoVE%22%5BJournal%5D)">HLA-Ig based artificial antigen presenting cells for efficient ex vivo expansion of human CTL.</a> Journal of Visualized Experiments : JoVE. (50), (2011).</li>
<li>Schütz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Killer+artificial+antigen-presenting+cells%3A+a+novel+strategy+to+delete+specific+T+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Killer artificial antigen-presenting cells: a novel strategy to delete specific T cells.</a> Blood. 111 (7), 3546-3552 (2008).</li>
<li>Snow, A. L., Pandiyan, P., Zheng, L., Krummey, S. M., Lenardo, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+power+and+the+promise+of+restimulation-induced+cell+death+in+human+immune+diseases%5BTitle%5D)+AND+%22Immunological+reviews%22%5BJournal%5D)">The power and the promise of restimulation-induced cell death in human immune diseases.</a> Immunological reviews. 236 (1), 68-82 (2010).</li>
<li>Schütz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+flow+cytometric+assay+for+the+simultaneous+analysis+of+antigen-specific+elimination+of+T+cells+in+heterogeneous+T+cell+populations%5BTitle%5D)+AND+%22Journal+of+immunological%22%5BJournal%5D)">A new flow cytometric assay for the simultaneous analysis of antigen-specific elimination of T cells in heterogeneous T cell populations.</a> Journal of immunological. 344 (2), 98-108 (2009).</li>
<li>Shen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((artificial+antigen-presenting+cells+deplete+alloantigen-specific+T+cells+in+a+murine+model+of+alloskin+transplantation%5BTitle%5D)+AND+%22Immunology+letters%22%5BJournal%5D)">artificial antigen-presenting cells deplete alloantigen-specific T cells in a murine model of alloskin transplantation.</a> Immunology letters. 138 (2), 144-155 (2011).</li>
<li>Perica, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanoscale+artificial+antigen+presenting+Cells+for+T+cell+immunotherapy%5BTitle%5D)+AND+%22Nanomedicine+nanotechnology%2C+biology%2C+and+medicine%22%5BJournal%5D)">Nanoscale artificial antigen presenting Cells for T cell immunotherapy.</a> Nanomedicine nanotechnology, biology, and medicine. 10 (1), 119-129 (2013).</li>
<li>Sunshine, J. C., Perica, K., Schneck, J. P., Green, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Particle+shape+dependence+of+CD8%2B+T+cell+activation+by+artificial+antigen+presenting+cells%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Particle shape dependence of CD8+ T cell activation by artificial antigen presenting cells.</a> Biomaterials. 35 (1), 269-277 (2013).</li>
</ol>

C.S. and M.F. have nothing to disclose.
Under a licensing agreement between NexImmune and the Johns Hopkins University, Drs. Schneck and Oelke are entitled to shares of royalty received by the University on sales of aAPC products described in this article. They also own NexImmune stock, which is subject to certain restrictions under University policy. Dr. Schneck is a member of the company&#34;s Board of Directors and Scientific Advisory Board. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.

The most critical step in the protocol is to ensure the appropriate ratio of signal 1 (peptide MHC) and signal 2 (anti-Fas mAb). We have performed intensive titration experiments to define the perfect ratio for signal 1 and 2; it became evident that minimal variations due to differences in the HLA-A2 Ig dimer or anti-Fas mAb concentrations can interfere with the functionality of the KaAPC. Thus, the concentration of both signals should always be verified and quality of both proteins should be frequently tested even if it was ordered from a commercial source. Furthermore, the concentrations should always be determined by the same assay as different detection assays might result in different concentrations. Functionality of the HLA-A2-Ig can also be impaired due to incomplete refolding and/or inefficient peptide loading. Furthermore dimer molecules can aggregate to different degrees, which could impact on the functional activity of the protein. Therefore, the actual amount of dimer needed for the generation of a functional and specific KaAPC may vary. In addition, we found that the ideal range for the death inducing signal anti-Fas mAb on KaAPC is extremely tight. In our hands amounts greater 3.64 &#956;g/ml lead to non-specific killing of Fas positive T cells while amounts below 3.64 &#956;g/ml resulted in a dose-depending reduced killing activity. While these conditions seem quite tight, attention to these details will result in the generation of a functional and specific KaAPC.
While the current KaAPC phenotype is functional for depletion of activated antigen-specific T cells, initial experiments targeting na&#239;ve or resting antigen-specific T cells demonstrated no significant induction of antigen-specific apoptosis as these populations have reduced Fas expression. Therefore, one might envision adding a co-stimulatory signal onto the KaAPC to induce the up-regulation of Fas on resting T cells to make turn them into a target for the KaAPC. While this is feasible all three signals will have to be carefully titrated to ensure a functional KaAPC phenotype.
Adjusting the bead platform to a biocompatible or biodegradable matrix will enhance KaAPC in vivo applicability. Recently, Shen et al. have reported the successful in vivo usage of KaAPC utilizing 5 &#956;m latex beads conjugated to anti-Fas (clone Jo2) and anti-His/H2-Kb-TRP211. We have been able to show that the general concept of artificial antigen-presenting Cells (aAPC) can be successfully transferred from &#956;m sized to nm sized particles12 and that functionality is influenced by particle geometry13. Thus, by varying the size and/or shape of future KaAPC designs one might be able to impact on the in vivo functionality and bio-distribution, which could be key for successful treatment of organ specific autoimmune diseases.
KaAPC overcome the major shortcomings of cell based depletion strategies as an easy to use &#8220;off the shelf&#8221;, time and cost efficient approach that is independent of donor condition and pre-treatment. KaAPC are not targets of self- or paracrine killing signaling and not sensitive to cytolytic effector functions of CTL. KaAPC will require identification of the relevant disease antigen and rely on their specific HLA type. While currently numerous suitable HLA-A2 restricted antigens for type 1 diabetes, GvHD and multiples sclerosis have been identified2, development of additional HLA class I dimer molecules as well as the ongoing identification of new autoimmune relevant antigens will further increase and broaden the applicability of KaAPC. Furthermore one could think of using KaAPC directed at different epitopes to target multiple antigens simultaneously.
In summary, KaAPC represent a flexible platform technology for antigen-specific T cell depletion. Their &#8220;lego-like&#8221; nature enables the inclusion of new signals such as additional co-stimulatory or inhibitory signals such as PD1 or TRAIL on various matrices, which could broaden and enhance their subsequent in vitro and in vivo applicability. We herein present the step-by-step protocol to generate KaAPC, the first bead based approach for the elimination of antigen-specific T cells from T cell mixtures with different specificities.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51859/51859fig3highres.jpg" width="500" /> 
Figure 3. KaAPC eliminate CTL in an antigen-specific fashion. PKH26-labelled activated Fas+ effector-memory CTL and PKH67-labelled CMVpp65-specific CTL from the same donor were mixed to a 1:1 ratio and co-cultured with CMVpp65KaAPC for 48 h. Control cultures were treated with unloadedKaAPC, or 1 &#181;g/ml of soluble anti-Fas-mAb (clone CH11). Minimal loss of viable cells was determined in untreated mixed CTL cultures (Tmix). Original FACS data for each CTL population of the Tmix are shown. Analysis of apoptosis was performed as previously published by separate gating on both T cell populations10. Numbers depicted in the upper left quadrants represents % of total cells. The illustrating figure is derived from one representative experiment out of three independent experiments. This research was originally published in Blood. Sch&#252;tz, C.&#160;et al. Killer artificial antigen-presenting cells: a novel strategy to delete specific T cells. Blood. 2008; 111, 3546&#8211;52. Copyright the American Society of Hematology. <a href="https://www.jove.com/files/ftp_upload/51859/51859fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Over the last two decades much progress has been made in understanding the pathogenic mechanisms of autoimmune diseases. However, the most common treatments still rely on corticosteroids as well as immunosuppressive drugs such as purine analogs, alkylating agents and calcineurin inhibitors1. The use of such drugs has greatly improved the prognosis of patients, yet the treatment does not provide a cure of the underlying immunological malfunction. Moreover, patients become vulnerable to infections and to the development of cancer.
Hence the ultimate goal for treatment of T cell mediated autoimmune diseases and allograft rejection is to specifically target only disease causing T cells while, at the same time, leaving the remaining immune system untouched and competent to fight immunological disorders. Utilizing antigen-presenting cells (APC) as a treatment to suppress peripheral T cell responses was first described as early as the 1970s by several groups. Since then many cell-based approaches have been developed (reviewed in Sch&#252;tz&#160;et al.2). Strategies using FasL-expressing killer-APC as immunoregulatory cells showed promising results indicating therapeutic potential for treatment of autoimmunity, allograft rejection and chronic infections. However, there are significant limitations with strategies utilizing FasL Killer-APC that have ultimately limited their clinical applicability; (i) generation or isolation of APC is time, labor and cost intensive (ii) patients suffering from cytopenia due to immunosuppression provide limited amounts and quality of cells (iii) coating or transduction of APC with apoptosis inducing signals such as FasL results in highly variable phenotypes and (iv) Killer-APC are sensitive to cytotoxic effector functions of T cells consequently reducing their therapeutic potential. Therefore, it is difficult to guarantee a defined reproducible Killer-APC phenotype that can be utilized for antigen-specific modulation of T cell responses in vivo.
Based on our previous work with FasL expressing Killer-APC3-5 and artificial antigen-presenting cells (aAPC)6,7 we developed a bead-based approach (KaAPC) for antigen-specific inhibition or elimination of auto-reactive T cells in vitro. KaAPC consist of a HLA-A2-Ig dimer (signal 1) and an anti-Fas mAb (apoptosis inducing signal) covalently immobilized onto a surface of a 4.5 &#956;m paramagnetic latex bead. They deplete antigen-specific T cells in a Fas/FasL mediated fashion from T cell cultures of multiple specificities in an antigen-specific manner and independent of the activation induced cell death (AICD) pathway. Dose dependent apoptosis in targeted T cells is induced rapidly already after 30 min8.
KaAPC can be easily generated in a controlled fashion ensuring a defined off the shelf phenotype and overcome most of the shortcomings of cell based depletion strategies. Consequently, KaAPC display great potential for treatment of T cell mediated autoimmune diseases and allograft rejection, advancing the field of T cell specific treatment strategies.

<table><tbody><tr><td>PBS</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Human AB serum</td><td>Atlanta biologicals</td><td>S40110</td><td></td></tr><tr><td>EDTA</td><td>Sigma/Aldrich</td><td>E5134</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma/Aldrich</td><td>71289</td><td></td></tr><tr><td>M-450 epoxybeads</td><td>Invitrogen/Dynal</td><td>14011</td><td></td></tr><tr><td>MPC-1 magnet</td><td>Invitrogen/Dynal</td><td>12001D</td><td></td></tr><tr><td>Screw top glass vial</td><td>Fisher Scientific</td><td>03-338AA</td><td></td></tr><tr><td>MACSmix Tube Rotator</td><td>Miltenyi</td><td>130-090-753</td><td></td></tr><tr><td>anti-CD95 (clone CH11)</td><td>Milliore</td><td>05-201</td><td></td></tr><tr><td>HLA-A2-Ig dimer</td><td>BD/Pharmingen</td><td>551263</td><td></td></tr><tr><td>Cryo-tube 2 ml</td><td>Corning</td><td>430488</td><td></td></tr><tr><td>anti-mouse IgG1 PE</td><td>BD/Pharmingen</td><td>559320</td><td></td></tr><tr><td>anti-mouse IgM FITC</td><td>BD/Pharmingen</td><td>553408</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck</td><td>S271-3</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma/Aldrich</td><td>C5080</td><td></td></tr><tr><td>HEPES</td><td>cellgro/Mediatech</td><td>25-060-CI</td><td></td></tr><tr><td>RPMI</td><td>gibco/life technologies</td><td>11875-085</td><td></td></tr><tr><td>96-well Round bottom plate</td><td>Falcon</td><td>353077</td><td></td></tr><tr><td>AnnexinV FITC</td><td>PromoKine</td><td>PK-CA577-1001-1000</td><td></td></tr><tr><td>7-AAD</td><td>BD/Pharmingen</td><td>51-68981E</td><td></td></tr><tr><td>FACS tubes</td><td>Falcon</td><td>352008</td><td></td></tr></tbody></table>

killer artificial antigen presenting cells (kaapc) for efficient in vitro depletion of human antigen-specific t cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

The features of this protocol include the defined generation of a KaAPC phenotype that displays HLA-A2-Ig (signal 1) and anti-Fas mAb (apoptosis signal) at the same time, which in comparison to cell based approaches is not altered by coating or transduction efficacies and can be easily modulated during production (Figure 1). Furthermore, KaAPC, are not sensitive to cytotoxic effector functions of T cells and their functionality is not dependent on the quality of autologous antigen-presenting cells pre and post in vitro manipulation.
KaAPC are capable of depleting antigen-specific T cells in vitro when loaded with cognate peptide, while non-cognate loaded KaAPC will not deplete T cells (Figure 2). Therefore, KaAPC induce T cell apoptosis in an antigen-specific manner. Subsequently, we used KaAPC for selective depletion of antigen-specific T cells from a mixture of different T cell specificities, demonstrating that only minimal cytotoxicity was leaking into neighbor T cells (Figure 3). Thus, KaAPC represent an exquisite tool for in vitro antigen-specific depletion of human activated antigen-specific T cells with potential clinical applicability for the treatment of autoimmune diseases and allograft rejections.

Watch this Scientific Journal Video about Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells at JoVE.com

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

killer-artificial-antigen-presenting-cells-kaapc-for-efficient-vitro

Research

JoVE Journal

Immunology and Infection

9.7K Views.  Johns Hopkins University.  Richtlijnen gepresenteerd voor het genereren van killer kunstmatige antigeenpresenterende cellen, KaAPC, een efficiënt hulpmiddel voor in vitro depletie van humane antigeen-specifieke T-cellen en een alternatieve oplossing voor cellulaire immunotherapie voor de behandeling van T-cel gemedieerde auto-immuunziekten in een antigen specifieke manier, zonder afbreuk te doen aan de resterende immuunsysteem. 

Video: Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular substrates offer key advantages over cell-based aAPC, including precise control of signal presentation parameters and physical properties of the aAPC surface to modulate its interactions with T cells. aAPC constructed from anisotropic particles, particularly ellipsoidal particles, have been shown to be more effective than their spherical counterparts at stimulating T cells due to increased binding and larger surface area available for T cell contact, as well as reduced nonspecific uptake and enhanced pharmacokinetic properties. Despite increased interest in anisotropic particles, even widely accepted methods of generating anisotropic particles such as thin-film stretching can be challenging to implement and use reproducibly.
To this end, we describe a protocol for the rapid, standardized fabrication of biodegradable anisotropic particle-based aAPC with tunable size, shape, and signal presentation for T cell expansion ex vivo or in vivo, along with methods to characterize their size, morphology, and surface protein content, and to assess their functionality. This approach to fabricating anisotropic aAPC is scalable and reproducible, making it ideal for generating aAPC for &#34;off-the-shelf&#34; immunotherapies.

Here, we present a protocol to quickly and reproducibly generate biologically inspired, biodegradable articifical antigen presenting cells (aAPC) with tunable size, shape, and surface protein presentation for T cell expansion ex vivo or in vivo.

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular ...

Environment

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Cancer Research

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of antigen-specific T cells, B) probe the dynamics of antigen-specific responses, C) understand how antigen-specific responses affect disease state such as autoimmunity, D) demystify heterogeneous responses for antigen-specific T cells, or E) utilize antigen-specific cells for therapy. The tool is based on a magnetic particle that we conjugate antigen-specific and T cell co-stimulatory signals, and that we term as artificial antigen presenting cells (aAPCs). Consequently, since the technology is simple to produce, it can easily be adopted by other laboratories; thus, our purpose here is to describe in detail the fabrication and subsequent use of the aAPCs. We explain how to attach antigen-specific and co-stimulatory signals to the aAPCs, how to utilize them to enrich for antigen-specific T cells, and how to expand antigen-specific T cells. Furthermore, we will highlight engineering design considerations based on experimental and biological information of our experience with characterizing antigen-specific T cells.

Antigen-specific T cells are difficult to characterize or utilize in therapies due to their extremely low frequency. Herein, we provide a protocol to develop a magnetic particle which can bind to antigen-specific T cells to enrich these cells and then to expand them several hundred-fold for both characterization and therapy.

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of ...

Engineering

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

Generation of Human Regulatory T Cell Clones

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as &#8220;Treg&#8221; therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4+CD25+ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

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